We have identified the RhoBTB2 putative tumor suppressor gene as a direct target of the E2F1 transcription factor. Overexpression of E2F1 led to upregulation of RhoBTB2 at the level of mRNA and protein. This also occurred during the induction of E2F1 activity in the presence of cyclohexamide, thus indicating that RhoBTB2 is a direct target. RNAi-mediated knockdown of E2F1 resulted in decreased RhoBTB2 protein expression—demonstrating that RhoBTB2 is a physiological target of E2F1. Since E2F1 primarily serves to transcribe genes involved in cell cycle progression and apoptosis, we explored whether RhoBTB2 played roles in either of these processes. We found RhoBTB2 expression highly upregulated during mitosis, which was partially dependent on the presence of E2F1. Furthermore, overexpression of RhoBTB2 induced a short-term increase in cell cycle progression and proliferation, while long-term expression had a negative effect on these processes. We similarly found RhoBTB2 upregulated during drug-induced apoptosis, with this being primarily dependent on E2F1. Finally, we observed that knockdown of RhoBTB2 levels via siRNA delayed the onset of drug-induced apoptosis. Collectively, we describe RhoBTB2 as a novel direct target of E2F1 with roles in cell cycle and apoptosis.